Lanthanide-binding properties of rat oncomodulin

Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2.     

Distance measurements between metal-binding sites of calmodulin and from these sites to Cys-133 of troponin I in the binary complex

Distances between the four Ca2+-binding sites of calmodulin (CaM) have been measured by fluorescence energy transfer techniques using Eu3+ and Tb3+ as energy donors and a number of other lanthanide ions (Ln3+) as acceptors. It was shown previously that lanthanide ions preferentially bind to sites I and II of CaM with an affinity higher than that for sites III and IV (Kilhoffer, M.-C., Demaille, J. G., and Gerald, D. (1980) FEBS Lett. 116, 269-272; Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., and Gergely, J. (1982) Eur. J. Biochem. 124, 7-12). Thus upon direct excitation with a laser the luminescence lifetimes of Eu1Ln1CaM and Tb1Ln1CaM provide information on the distance between sites I and II. On the other hand, since Tb3+ ions bound to sites III and IV are sensitizable through tyrosine residues, lifetime measurements of Tb2Ln2CaM excited by UV light yield the distance between sites III and IV. Both pairs of sites were found to be separated by a distance of 1.05 +/- 0.07 nm. Binding of Ca2+ to sites III and IV does not alter the distance between sites I and II. We have also attached a chromophoric label, dimethylaminophenylazobenzene, to Cys-133 of skeletal troponin I and carried out distance measurements on its complex with CaM by both direct and indirect excitation. The averaged distances from sites I and II in the N-terminal half and from sites III and IV in the C-terminal half of the CaM molecule to the label on troponin I are 2.7 and 2.5 nm, respectively.     

Spectroscopic studies of metal ion binding to a tryptophan-containing parvalbumin

The binding of Ca(II) and members of the trivalent lanthanide ion, Ln(III), series to apoparvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) results in the development of a distinctive sharp feature in the UV absorption spectrum at about 290 nm. Titration curves obtained by monitoring the spectral change in this region reveal a change in slope after the addition of 1 equiv of metal ion and no further rise after 2 equiv has been added, consistent with sequential binding to the principal EF and CD sites. Laser-induced luminescence excitation spectra of the 7F0----5D0 transition of bound Eu(III) demonstrate the quantitative binding of this ion to the principal sites and disclose the presence of a subsidiary site at pH values greater than 6. Metal ion competition experiments monitored by means of this excitation transition show that the early members of the Ln(III) ion series bind more tightly than those at the end. Tryptophan-sensitized Tb(III) luminescence reveals that this ion binds sequentially to the EF and CD sites, in that order. The intrinsic tryptophan fluorescence of apoparvalbumin is increased in a stepwise fashion as Ca(II) or Ln(III) ions bind sequentially, with the exceptions of Eu(III) and Yb(III). The binding of the latter two ions causes quenching of the protein fluorescence via an energy-transfer process which involves low-lying charge-transfer bands. The distance dependences of the tryptophan to Tb(III) and tryptophan to Eu(III) energy-transfer processes are observed to be identical, consistent with a F?rster-type mechanism in both cases.     

Stopped-flow kinetic studies of metal ion dissociation or exchange in a tryptophan-containing parvalbumin

The rates of dissociation of 2 equiv of various metal ions [Ca(II), Cd(II), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Yb(III), and Lu(III)] from the primary CD and EF metal ion binding sites of parvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) were measured by stopped-flow techniques. The removal or replacement of metal ions was monitored by changes in sensitized Tb(III) luminescence or in intrinsic protein tryptophan fluorescence as quenching ions [Eu(III) or Yb(III)] were bound or removed or as the apoprotein was formed. In experiments wherein the bound metal ions were removed by mixing the parvalbumin with an excess of 1,2-diaminocyclohexanetetraacetic acid (DCTA), the kinetic traces were best fit by a double exponential with koff rate constants of 1.07 and 5.91 s-1 for Ca(II), 1.54 and 10.5 s-1 for Cd(II), and approximately 0.05 and approximately 0.5 s-1 for all of the trivalent lanthanide ions. In experiments wherein the bound metal ions were exchanged with an excess of a different metal ion, pseudo-first-order rate constants were proportional to the concentration of excess attacking metal ion for both the fast and slow processes in most experiments. In these cases, extrapolation of the rate constants to zero concentration of attacking metal ion gave values which agree well with the DCTA scavenging results. This finding demonstrates that the off rate constants do not depend on the occupancy of the neighboring site and therefore implies that there is no significant cooperativity in metal ion binding between the two sites in parvalbumin.     

Europium(III) luminescence and tyrosine to terbium(III) energy-transfer studies of invertebrate (octopus) calmodulin.

The effects of minor differences in the amino acid sequences between a vertebrate (bovine testes) and an invertebrate (octopus) calmodulin on metal ion binding were investigated via laser-induced Eu3+ and Tb3+ luminescence. Amino acid substitutions at residues which are coordinated to the metal ion do not produce any detectable changes in the 7F0----5D0 excitation spectrum of the Eu3+ ion bound to octopus calmodulin relative to bovine testes calmodulin; only minor differences in the excited-state lifetime values in D2O solution are observed. The dissociation constants for Eu3+ (1.0 +/- 0.2 microM) and Tb3+ (5 +/- 1 microM) from the weak lanthanide binding sites (III and IV, numbered from the amino terminus) of octopus calmodulin were measured using luminescence techniques. Both values agree well with those reported previously for bovine testes calmodulin [Mulqueen, P. M., Tingey, J. M., & Horrocks, W. D., Jr. (1985) Biochemistry 24, 6639-6645]. The measured dissociation constant of Eu3+ bound in the tight lanthanide binding sites (I and II) is 6 +/- 2 nM for octopus calmodulin and 12 +/- 2 nM for bovine testes calmodulin. The distances between sites I and II (12.4 +/- 0.5 A) and sites III and IV (11.7 +/- 0.8 A) were determined from F?rster-type energy transfer in D2O solutions of octopus calmodulin containing bound Eu3+ donor and Nd3+ acceptor ions. F?rster theory parameters for nonradiative energy transfer between Tyr138 and Tb3+ ions bound at sites III and IV of octopus calmodulin were comprehensively evaluated, including a dynamics simulation of the orientation factor kappa 2. This theory is found to account quantitatively for the observed energy-transfer efficiency as evaluated from the observed sensitized Tb3+ emission.     

Probing the metal-binding sites of cod parvalbumin using europium(III) ion luminescence and diffusion-enhanced energy transfer.

Excitation spectroscopy of the 7F0----5D0 transition of Eu3+ and diffusion-enhanced energy transfer are used to study metal-binding characteristics of the calcium-binding protein parvalbumin from codfish. Energy is transferred from Eu3+ ions occupying the CD- and EF-binding sites to the freely-diffusing Co(III) coordination complex energy acceptors: [Co(NH3)6]3+, [Co(NH3)5H2O]3+, [CoF(NH3)5]2+, [CoCl(NH3)5]2+, [Co(NO2)3(NH3)3], and [Co(ox)3]3-. In the absence of these inorganic energy acceptors, the excited-state lifetimes of Eu3+ bound to the CD and EF sites are indistinguishable, even in D2O; however, in the presence of the positively charged energy acceptor complexes, the Eu3+ probes in the cod parvalbumin have different excited-state lifetimes due to a greater energy-transfer site from Eu3+ in the CD site than from this ion in the EF site. The observation of distinct lifetimes for Eu3+ in the two sites allows the study of the relative binding site affinities and selectivity, using other members of the lanthanide ion series. Our results indicate that during the course of a titration of the metal-free protein, Eu3+ fills the two sites simultaneously. Eu3+ is competitively displaced by other Ln3+ ions, with the CD site showing a preference for the larger Ln3+ ions while the EF site shows little, if any, competitive selectivity across the Ln3+ ion series.     

Steady-state luminescence investigation of the binding of Eu(III) and Tb(III) ions with synthetic peptides derived from plant thionins

This work reports Eu(III) and Tb(III) luminescence titrations in which the lanthanide ions were used as spectroscopic probes for Ca(II) ions to determine the metal binding ability of Ac-NESVKEEGGW-NH(2) and Ac-NESVKEDGGW-NH(2). These decapeptides correspond to the putative calcium binding region of the plant antifungal proteins SI-alpha1 from Sorghum bicolor and of Zeathionin from Zea mays, respectively. The luminescence spectra for the Eu(III)-decapeptide system (red emission) with the excitation at the Trp band at 280 nm showed an enhancement of the intensities of the 5D(0)-->7F(J) transitions (where J=0-4) with increments of Eu(III) ion concentration. The photoluminescence titration data of the terbium ion (green emission) in the decapeptide solutions showed intensification of the 5D(4)-->7F(J) transitions (J=0-6), similar to that observed for the Eu(III) ion. Thus, energy transfer from Ac-NESVKEEGGW-NH(2) and Ac-NESVKEDGGW-NH(2) to the trivalent lanthanide ions revealed that these peptides are capable of binding to these metal ions with association constants of the order of 10(5) M(-1). The amino acid derivative Ac-Trp-OEt also transferred energy to Tb(III) and Eu(III) ions as judged from the quenching of tryptophan luminescence. However, the energy transfers were significantly lower. Taken together the luminescence titration data indicated that Ac-NESVKEEGGW-NH(2) and Ac-NESVKEDGGW-NH(2) bind efficiently to both trivalent lanthanide ions and that these ions may be used as probes to distinguish an anionic peptide from a neutral amino acid derivative.     

Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin

The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.     

Chemiluminescence and energy transfer mechanism of lanthanide ions in different media based on peroxomonosulfate system

The chemiluminescence (CL) phenomena of lanthanide (Ln) ions and their coordinate complexes in peroxomonosulfate system and the energy transfer mechanism during the process were investigated in this work. A strong and sharp CL signal was yielded when the Eu(III) or Tb(III) solution was added to the peroxymonosulfate solution. The CL intensity was greatly enhanced by 2,6‐pyridinedicarboxylic acid (DPA) ligand [maximum enhancement reached when Ln(III):DPA was 1:1] and hexadecyltrimethylammonium chloride micelles. The degree of enhancement of DPA and micelles on Ln(III) CL was related to the fluorescence lifetimes of Ln(III) in different media. According to the ESR spin‐trapping experiments of 2,2,6,6‐tetramethyl‐4‐piperidone and the specific quenching experiments of 1,4‐diazabicyclo[2.2.2]octane and sodium azide, singlet oxygen was generated though the Ln(III) ion‐catalyzed decomposition of peroxymonosulfate. From the comparisons of the fluorescence and CL spectra, lanthanide ions were the luminescence emitter and the ligand DPA absorbed the energy from singlet oxygen and transferred it to Ln(III) ions in the coordinate complexes. Micelles can enhance the CL intensity by improving intermolecular energy transfer efficiencies, removing the quenching effect of water and prolonging the lifetime of singlet oxygen. Copyright © 2009 John Wiley & Sons, Ltd.     

Lanthanide spectroscopic studies of the dinuclear and Mg(II)-dependent PvuII restriction endonuclease

Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10-20 microM), Eu(III) excitation (7)F(0) --> (5)D(0) spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 microM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (K(d) = 1-3 microM) and a much weaker site (K(d) approximately 100-200 microM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 microM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function.     

Circularly polarized luminescence from terbium(III) as a probe of metal ion binding in calcium-binding proteins.

A number of different experimental techniques have been used to probe the details of structural changes on the binding of Ca(II) to the large number of known calcium-binding proteins. The use of luminescent lanthanide(III) ions, especially terbium(III) and europium(III), as substitutional replacement for calcium(II), has led to a number of useful experiments from which important details concerning the metal ion coordination sites have been obtained. This work is concerned with the measurement of the circularly polarized luminescence (CPL) from the 5D4----7F5 transition of Tb(III) bound to the calcium binding sites of bovine trypsin, bovine brain calmodulin, and frog muscle parvalbumin. It is demonstrated that it is possible to make these polarization measurements from very dilute solutions (less than 20 microM) and monitor structural changes as equivalents of Tb(III) are added. It is shown that the two proteins that belong to the class of "EF-hand" structures (calmodulin and parvalbumin) possess quite similar CPL line shapes, whereas Tb(III) bound to trypsin has a much different band structure. CPL results following competitive and consecutive binding of Ca(II) and Tb(III) bound to calmodulin are also reported and yield information concerning known differences between the sequence of binding of these two species.     

Metal ion binding sites of bacteriorhodopsin. Laser-induced lanthanide luminescence study

Laser-excited luminescence lifetimes of lanthanide ions bound to bacteriorhodopsin have been measured in deionized membranes. The luminescence titration curve, as well as the binding curve of apomembrane (retinal-free) with Eu3+, has shown that the removal of the retinal does not significantly affect the affinity of Eu3+ for the two high affinity sites of bacteriorhodopsin. The D2O effects on decay rate constants indicate that Eu3+ bound to the high affinity sites of native membrane or apomembrane is coordinated by about six ligands in the first coordination sphere. Tb3+ is shown to be coordinated by four ligands. The data indicate that metal ions bind to the protein with a specific geometry. From intermetal energy transfer experiments using Eu3+-Pr3+, Tb3+-Ho3+, and Tb3+-Er3+, the distance between the two high affinity sites is estimated to be 7-8 A.     

Intermolecular chiral recognition processes probed by chiroptical luminescence

Enantiopreferential energy transfer processes between dissymmetric lanthanide and transition metal complexes dissolved in acetonitrile are studied using chiroptical luminescence techniques. The energy donors (luminophores) in this study are a racemic mixture of Ln(dpa)3 (3-) complexes (where Ln = Eu3+ or Tb3+ and dpa = 2,6-pyridinedicarboxylate), and the energy acceptors (quenchers) are an enantiomerically-resolved population of Co(R,R-chxn)3 3+ (where R,R-chxn = trans-1R,2R-diaminocyclohexane) complexes. The luminophores are dissolved in acetonitrile as (NEt4)3[Ln(dpa)3] (where NEt(4) = tetraethlylammonium) and (NBu4)[Ln(dpa)3] (where NBu4 = tetrabutylammonium) salts. The unquenched luminescence lifetimes are reported for both Eu(dpa)3 (3-) and Tb(dpa)3 (3-) in acetonitrile over the range 263-333 K, and these results are compared to luminescence lifetimes in aqueous solution. Time-resolved chiroptical luminescence measurements of enantiopreferential quenching kinetics are reported for samples with Eu(dpa)3 (3-) and Co(R,R-chxn)3 3+ in acetonitrile over 263-333 K range. These results are analyzed using a phenomenological quenching kinetics model, and the results are compared to results in aqueous solution. These comparisons show that the overall Eu-Co luminescence quenching efficiency is reduced in acetonitrile vs. aqueous samples, because the salts of (NX4)3[Eu(dpa)3] are not completely dissociated in acetonitrile. However, the enantiopreference exhibited is identical in acetonitrile vs. aqueous solution.     

Metal-ion binding to parvalbumin. A 113Cd-n.m.r. study of the binding of different lanthanide ions.

113Cd-n.m.r. studies were used to investigate the binding of the lanthanide ions La3+, Gd3+, Tb3+, Yb3+ and Lu3+ to parvalbumins. It was shown that lanthanide ions with a smaller ionic radius bind sequentially to Cd2+-saturated parvalbumin, whereas those with a larger ionic radius bind with similar affinity to both the CD site and the EF site. The smallest ion, Lu3+, does in fact not compete significantly with Cd2+ for the CD site in carp parvalbumin, but appears to bind only to the EF site. This preference of the smaller lanthanide ions for the EF site was used to assign the n.m.r. signals for protein-bound 113Cd. By using Cd n.m.r. and Tb3+ fluorescence it was also shown for alpha-lineage parvalbumin from pike that these proteins possess a third site that can bind lanthanide ions. This site is, however, much weaker than in the beta-lineage parvalbumins. It was used to assign the 113Cd resonances from protein-bound Cd2+ ions in the spectrum of pike pI5.0 parvalbumin.     

Calcium binding proteins: optical stopped-flow and proton nuclear magnetic resonance studies of the binding of the lanthanide series of metal ions to parvalbumin

Optical stopped-flow techniques have been used to determine the dissociation rate constants (koff) for the lanthanide(III) ions from carp (pI 4.25) parvalbumin. For most of the 13 different lanthanides studied, the release kinetics were diphasic, composed of both a fast phase (whose rate varied across the series, La3+ leads to Lu3+, between the limits -1.2 less than or equal to log kFAST less than or equal to -0.7) and a slower phase (whose rate varied across the series, La3+ leads to Lu3+, between the limits -1.2 greater than or equal to log kSLOW greater than or equal to -2.9). In addition, the La3+- and Lu3+-induced changes in the 270-MHz proton nuclear magnetic resonance spectrum of parvalbumin were used to calculate the dissociation constants for these specific lanthanides from the two high-affinity Ca2+ binding sites. The KD for one site appears to remain constant across the lanthanide series, determined to be 4.8 X 10(-11) M for both La3+ and Lu3+. The other site, however, is evidently quite sensitive to the nature of the bound Ln3+ ion and shows a strong preference for La3+ (KD,La = 2.0 X 10(-11) M; KD,Lu = 3.6 X 10(-10) M). We conclude from these observations that reports of nearly indistinguishable CD/EF binding site affinities for parvalbumin complexes of the middle-weight lanthanides (i.e., Eu3+, Gd3+, and Tb3+) are quite reasonable in view of the crossover in relative CD/EF site affinities across the lanthanide series.     

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Non-cooperative Ca(II) removal and terbium(III) substitution in carp muscle calcium binding parvalbumin.

Close coorelation of atomic absorption measurements for Ca(II) contents indicates that from pH 5.8-7.4 a twentyfold excess of EGTA1 removes but one of two Ca(II) from carp parvalbumin. Thus binding of the two Ca(II) appears to be noncooperative. The maximum in emission intensity observed at a nonintegral 1.4-1.7 equivs of added Tb(III) is shown to be due to quenching by excess Tb(III). The emission intensity at the maximum increased 40% upon dialysis to remove Tb(III) not bound in the CD or EF sites. Atomic absorption results show that both Ca(CD) and Ca(EF) of native parvalbumin are easily replaced by Tb(III). Emission of Tb(EF) is not quenched by Tb(CD), but by solution Tb(III) bound at a third site, perhaps the single water molecule bound to Tb(EF). Labeling of the single sulfhydryl group with a trifluoroacetonyl gorup yields a protein with ultraviolet circular dichroism, emission, and circularly polarized emission spectra closely similar to those of native parvalbumin.     

Binding of europium(III) ions to RNA stem loops: role of the primary hydration sphere in complex formation

Understanding the process by which RNA molecules fold into stable structures includes study of the role of site-bound metal ions. Because the alkaline earth metal ions typically associated with RNA structure [most often Mg(II)] do not provide convenient spectroscopic signals, replacement with metal ions having spectroscopically useful properties has been a valuable approach. The luminescence properties of the lanthanide(III) series, in particular europium(III), have made them useful in the study of complexation with biomolecules. We review the physical, chemical, and spectroscopic characteristics of Eu(III) that contribute to its value as a probe of RNA-metal ion interactions, and examples of information obtained from studies of Eu(III) bound to small RNA stem loops. Although Eu(III) has similar site preference to Mg(II), luminescence and isothermal titration calorimetry measurements indicate that Ln(III) loses water molecules from the inner hydration sphere more readily than does Mg(II), resulting in more direct coordination between RNA and the metal ion and very different energetics of binding. In some cases, e.g., a GAAA tetraloop, binding appears to occur by a lock and key process; in the same base sequence containing certain deoxynucleoside substitutions that alter loop structure, binding appears to occur by an induced fit process.     

Interaction of parvalbumin of pike II with calcium and terbium ions

Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from F?rster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.     

High-resolution proton and laser photochemically induced dynamic nuclear polarization NMR studies of cation binding to bovine alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) is a calcium binding protein that also binds Mn(II), lanthanide ions, A1(III), Zn(II), Co(II). The structural implications of cation binding were studied by high-resolution proton (200 MHz) NMR and photochemically induced dynamic nuclear polarization (CIDNP) spectroscopy. Marked changes were observed in the NMR spectra of the apoprotein upon addition of a stoichiometric amount of calcium to yield Ca(II)-alpha-LA, manifested particularly in ring current shifted aliphatic peaks and in several shifts in the aromatic region, all of which were under slow exchange conditions. The CIDNP results showed that two surface-accessible tyrosine residues, assigned as Tyr-18 and -36, became inaccessible to the solvent upon addition of 1:1 Ca(II) to apo-alpha-lactalbumin, while Tyr-103 and Trp-104 remained completely accessible in both conformers. The proton NMR spectra of apo-alpha-LA and A1(III)-alpha-LA were extremely similar, which was also consistent with intrinsic fluorescence results [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. The paramagnetic cation Mn(II) bound to the strong calcium binding site on apo-alpha-LA but also to the weak secondary Ca(II) binding site(s) on Ca(II)-alpha-LA. It was also found that Co(II) bound to some secondary sites on Ca(II)-alpha-LA that overlapped the weak calcium site. All of the lanthanide shift reagents [Pr(III), Eu(III), Tb(III), Dy(III), Tm(III), Yb(III)] bound under slow exchange conditions; their relative affinities for apo-alpha-lactalbumin from competitive binding experiments were Dy(III), Tb(III), and Pr(III) greater than Ca(II) greater than Yb(III).